Project Summary/Abstract The objective of this Phase I STTR application is to develop a next generation quantitative drug resistance assay that can accurately determine the abundance and linkage of HCV variants resistant to direct-antiviral agents (DAAs). Drug resistance to HCV is a major threat to achieving sustained virologic response (SVR) in HCV-infected individuals. RAVs pre-exist in patients nave to DAA therapy, and resistant variants are selected after treatment failures. Presence of HCV resistance-associated variants (RAVs) is known to impact the efficacy of DAAs. The FDA has now recommended baseline resistance testing prior to initiation of certain DAAs regimens. In addition, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) have recommended resistance testing in patients failing initial DAAs to guide the selection of re-treatment regimens. Emerging data from clinical trials suggest that the identity and the abundance of RAVs may impact treatment outcome. Furthermore, linkage of RAVs may compromise clinical response to DAAs. However, commercially available HCV resistance assays cannot accurately determine the abundance or linkage of RAVs. Dr. Wang's laboratory recently developed a quantitative Single Variant Sequencing (qSVS) assay, which takes advantage of the speed and accuracy of the high-throughput MiSeq technology, and a random sequencing tags strategy that removes biases and technical artifacts known to obscure true representations of sequence variants. By developing a bioinformatics pipeline that automates analysis of drug resistance calls, his laboratory has applied this qSVS approach to quantify HCV RAVs from in vitro and clinical samples. Medosome Biotec and its research partner at UF hypothesize that this qSVS approach can be optimized to quantify sub-populations of drug resistant HCV accurately. We will test this hypothesis by pursuing two Specific Aims: 1) Optimize the qSVS method for accurate quantification of HCV NS3, NS5A, and NS5B drug resistance variants, and 2) Conduct initial testing of the optimized qSVS method using HCV plasmids/replicons and clinical isolates. The qSVS approach is innovative because it will enable a paradigm shift in precision medicine by leveraging the random sequencing tag method and the speed, accuracy, and the long paired-end read capability of MiSeq personal sequencer to produce a new resistance assay for quantitative detection of HCV drug resistant variants. If successful, the qSVS assay will be validated and developed in Phase II studies. An accurate and low cost qSVS assay will have tremendous commercialization potential, given the global burden of HCV with millions of HCV-infected individuals who will require resistance testing to maximize treatment success during DAA therapy.